Biology subjects

Brewster, J. L.

Publications and source records attributed to Brewster, J. L..

4 recordsLinked to original sources

bioRxiv2022

Structure of phage lambda Red-beta(177) annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination

The bacteriophage {lambda} red recombination system catalyzes the single-strand annealing homologous DNA recombination reaction, in which Red{beta} annealase protein plays a critical role. Using cryogenic electron microscopy, we were able to determine a structure of a C-terminally truncated Red{beta} with the residues 1-177 bound to two complementary 27mer oligonucleotides forming an annealing intermediate, to a final resolution of 3.3 [A]. This structure validates and rationalizes decades of experimental observations on the biochemistry of Red{beta}. Definition of the interaction surfaces between subunits explains not only the DNA binding properties of Red{beta}, but also its propensity to oligomerize into long helical filaments, which are also formed by almost all annealases and are known to be functionally important. In addition, this annealing intermediate structure provides a detailed picture of the hydrogen bonding network that positions the DNA strands in a planar orientation to facilitate base pairing. Residues 133-138, which are missing from our structure, form a flexible loop. Molecular dynamics simulations were used to model the range of motion of the flexible loop, which suggested that it has a crucial role in keeping the DNA strands in the DNA-binding groove long enough to allow homology searching. The combination of structural and computational observations has allowed us to propose a detailed mechanism for the action of Red{beta}. More than half a century after its discovery, our work shines a light not only on the structure and mechanisms of Red{beta}, but also of other proteins within the annealase superfamilies. Significance StatementSingle-strand annealing homologous DNA recombination is a process that is conserved throughout evolution from bacteriophages to humans, highlighting its importance and indispensability. It is a process that repairs double-stranded DNA breaks and is particularly vital in dsDNA viruses. The Red{beta} protein from the bacteriophage lambda is the archetypal annealase protein, forming the basis of our knowledge about this class of proteins. Along with the exonuclease {lambda}Exo, these two proteins not only form the model system for single-strand annealing homologous recombination, but are also used in thousands of laboratories worldwide for performing genetic manipulations. After its discovery in 1966, we report the first structure of the DNA-binding and oligomerization domain of Red{beta}, providing details about the mechanism of homologous DNA annealing.

biochemistry↗

bioRxiv2020

Structural basis for active-site probes targeting Staphylococcus aureus serine hydrolase virulence factors

Staphylococcus aureus is a major cause of infection in the community and in hospitals. Serine hydrolases play key roles in bacterial homeostasis, in particular biofilms. Activity-based profiling has previously identified a family of serine hydrolases, designated fluorophosphonate-binding hydrolases (Fphs), which contribute to virulence of S. aureus in the biofilm niche. Here we report structures of the putative tributyrin esterase FphF, alone and covalently bound by a substrate analog, and small molecule inhibitors that occupy the hydrophobic substrate-binding pocket. We show that FphF has promiscuous esterase activity. Building from this, we extended our analysis to the wider Fph protein family using homology modeling and docking tools. We predict that other Fph enzymes, including FphB which was linked directly to virulence, may be more specific than FphF. This study provides insight into Fph function and a template for designing new imaging agents, diagnostic probes, and inhibitors to treat S. aureus infections.

biochemistry↗

bioRxiv2019

Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

Apoptosis signal-regulating kinases (ASK1-3) are activators of the P38 and JNK MAP kinase pathways. ASK1-3 form oligomeric complexes known as ASK signalosomes that initiate signalling cascades in response to diverse stress stimuli. Here we demonstrate that oligomerization of ASK proteins is driven by previously uncharacterised sterile-alpha motif (SAM) domains that reside at the C-terminus of each ASK protein. SAM domains from ASK1-3 have distinct behaviours: ASK1 forms unstable oligomers, ASK2 is predominantly monomeric, and the ASK3 SAM domain forms a stable oligomer even at low concentration. In contrast to their isolated behaviour, the ASK1 and ASK2 SAM domains preferentially form a stable heterocomplex. The crystal structure of the ASK3 SAM domain, small-angle X-ray scattering, and mutagenesis suggests that ASK3 oligomers and ASK1-ASK2 complexes form discrete quasi-helical rings, via the mid-loop-end-helix interface. Preferential ASK1-ASK2 binding is consistent with mass spectrometry showing that full-length ASK1 forms heterooligomeric complexes incorporating high levels of ASK2. Accordingly, disruption of SAM domain-association impairs ASK activity in the context of electrophilic stress induced by 4-hydroxy-2-nonenal. These findings provide a structural template for how ASK proteins assemble foci to drive inflammatory signalling, and reinforce that strategies targeting ASK kinases should consider the concerted actions of multiple ASK family members.

biochemistry↗

bioRxiv2018

Substrate binding allosterically relieves autoinhibition of the TRIB1 pseudokinase

One Sentence SummarySubstrate binding to Tribbles-homolog 1 (TRIB1) pseudokinase induces allosteric changes that allow formation of a complex with the COP1 ubiquitin ligase.\n\nAbstractThe Tribbles family of pseudokinases recruit substrates to the COP1 ubiquitin ligase for ubiquitination. CCAAT-enhancer binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid development. Here we show that the TRIB1 pseudokinase can recruit various C/EBP family members, with binding of C/EBP{beta} attenuated by phosphorylation. To explain the mechanism of substrate recruitment, we solved the crystal structure of TRIB1 in complex with C/EBP. TRIB1 undergoes a significant conformational change relative to its substrate-free structure, to bind C/EBP in a pseudo-substrate-like manner. Crucially, substrate binding triggers allosteric changes that link substrate recruitment to COP1 binding, which is consistent with molecular dynamics and biochemical studies. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation-- via the activation loop and C-helix--and raise the possibility of small molecules targeting either the activation loop-in, or loop-out, conformations of Tribbles pseudokinases.

biochemistry↗